Periodic Reporting for period 1 - HDACbyCRYOEM (High-resolution cryo-EM structures of the human and yeast Sin3 histone deacetylase complexes)
Période du rapport: 2022-10-01 au 2024-09-30
Histone deacetylase (HDAC) complexes are the main transcriptional repression machineries in eukaryotes. Their disruption leads to gene expression deregulations and causes numerous diseases, involving cancers, inflammatory diseases, and neurological disorders. The 11 promiscuous HDAC enzymes in human have thus been widely exploited to study fundamental transcription-related mechanisms and deliver small-molecule therapeutics, including five HDAC-inhibiting drugs that have been approved by the US Food and Drug Administration (FDA) . Owing to their lack of specificity and toxicity in vivo, current HDAC inhibitors have been limited to the treatment of haematological cancers. However, HDAC complexes are made of multiple other protein subunits that are poorly characterized. Better understanding of HDAC complex biology would thus lead to the development of more specific inhibitors and treatments focused on alternative protein targets.
In this context, the absence of complete, high-resolution 3D structures for the whole SIN3 HDAC machineries has hindered the precise understanding and validation of how small molecules can perturb the overall architecture of the complexes to reprogram their biological functions. This has also precluded chemical optimizations of the initial molecules and design of novel compounds targeting different proteins. In this application, we proposed to combine the expertise of the applicant in HDAC complex biology and druggability, to that of his supervisor in structural biology and cryo-EM to solve the structures of: 1) the human SIN3A HDAC complex [specific aim 1], and 2) the highly-conserved yeast S. cerevisiae Sin3/Rpd3L HDAC complex [specific aim 2], at near atomic resolution, by single-particle cryogenic electron microscopy (cryo-EM).
In this context, the absence of complete, high-resolution 3D structures for the whole SIN3 HDAC machineries has hindered the precise understanding and validation of how small molecules can perturb the overall architecture of the complexes to reprogram their biological functions. This has also precluded chemical optimizations of the initial molecules and design of novel compounds targeting different proteins. In this application, we proposed to combine the expertise of the applicant in HDAC complex biology and druggability, to that of his supervisor in structural biology and cryo-EM to solve the structures of: 1) the human SIN3A HDAC complex [specific aim 1], and 2) the highly-conserved yeast S. cerevisiae Sin3/Rpd3L HDAC complex [specific aim 2], at near atomic resolution, by single-particle cryogenic electron microscopy (cryo-EM).
To achieve our first objective, a new method was developed to purify the Sin3/Rpd3L HDAC complex from yeast S. cerevisiae. We benchmarked and optimized the tandem affinity purification (TAP) procedure to produce highly pure, fully-assembled, and high-yield samples suitable for biochemical/biophysical assays, mass spectrometry, and cryo-EM studies. The purified complexes exhibited the expected protein composition, minimal contaminations, and an average particle radius of about 14 nanometers. After stabilizing the complex by cross-linking, the sample was applied to microscope grids and Cryo-EM imaging was performed. We observed a good particle distribution on the grid and the collected data yielded a structure of the complex at a resolution of 3.37 Å. Importantly, results were independently repeated at least three times and structures at comparable resolutions (i.e. around 3.5 Å) were obtained in each case; showing the reproducibility of the established cryo-EM pipeline. To further leverage our findings, we are exploring the binding of the Sin3/Rpd3L HDAC complex to inhibitors and specific nucleosome substrates. Using our optimized protocol, we have already obtained a cryo-EM structure of the complex bound to an HDAC inhibitor (~3.5 Å), which revealed the binding mode in a multiprotein complex environment. Overall, these findings demonstrate the robustness of the protein purification/cryo-EM platform developed at the host institution during the MSCA project.
For the second objective, a human SIN3A HDAC complex (containing dozens of subunits) was purified using an established tandem affinity purification method in human cells. Cells were transfected with a plasmid co-expressing the tagged SUDS3 and SIN3A subunits, which were then captured using affinity beads. The purified complex contained expected components, with mass spectrometry analysis showing 80% purity, suitable for further studies. Samples and buffers were optimized to be compatible with cryo-EM studies and data collections on different purified fractions are expected to produce results in the near future.
For the second objective, a human SIN3A HDAC complex (containing dozens of subunits) was purified using an established tandem affinity purification method in human cells. Cells were transfected with a plasmid co-expressing the tagged SUDS3 and SIN3A subunits, which were then captured using affinity beads. The purified complex contained expected components, with mass spectrometry analysis showing 80% purity, suitable for further studies. Samples and buffers were optimized to be compatible with cryo-EM studies and data collections on different purified fractions are expected to produce results in the near future.
For the first objective, a major achievement was to show that the purified Sin3/Rpd3L HDAC complex can bind to specific nucleosome substrates. We expect to obtain a cryo-EM structure to reveal the molecular details of the binding mechanisms in the near future, which will allow the scientific community to better understand, for example, promoter recognition by HDAC complexes. In addition, the rewiring of HDAC complexes following inhibitor bindings will help the community to better design the next generation of HDAC-targeting compounds.
For the second objective, the cryo-EM structures of the human SIN3A HDAC complex we expect to acquire in the near future will be the first of their kinds. They will allow us to reveal conserved mechanisms and architectures between yeast and human cells, in addition to provide novel interfaces for the rationale design of novel compounds and treatment options. In particular, this will be greatly facilitated by the optimized purification protocol we developed during the two years of MSCA funding.
For the second objective, the cryo-EM structures of the human SIN3A HDAC complex we expect to acquire in the near future will be the first of their kinds. They will allow us to reveal conserved mechanisms and architectures between yeast and human cells, in addition to provide novel interfaces for the rationale design of novel compounds and treatment options. In particular, this will be greatly facilitated by the optimized purification protocol we developed during the two years of MSCA funding.